Method for producing lysine derivative

ABSTRACT

The present invention provides a method for industrially producing an optically active lysine derivative useful as a pharmaceutical intermediate. More particularly, the present invention provides a production method including protecting an amino group or an amino group and carboxyl group of optically active 2-amino-6-methyl-6-nitroheptanoic acid with a protecting group, reducing a nitro group to synthesize a 6,6-dimethyl lysine derivative and reacting the 6,6-dimethyl lysine derivative with an acetic acid derivative.

This application is a division of application Ser. No. 10/281,977 filedOct. 29, 2002 now U.S. Pat. No. 6,664,412 which is a continuation ofapplication Ser. No. 09/903,531 filed Jul. 13, 2001 abandoned.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method for producing a specific,optically active lysine derivative useful as a pharmaceuticalintermediate.

BACKGROUND OF THE INVENTION

Optically active lysine derivatives, such as an optically active6,6-dimethyl lysine derivative of the formula (3)

wherein * means an asymmetric carbon atom, P₁ and P₂ are eachindependently an amino-protecting group or hydrogen atom where P₁ and P₂are not hydrogen atoms at the same time, or P₁ and P₂ in combinationshow an amino-protecting group, and P₄ is a hydrogen atom orcarboxyl-protecting group, and an optically active lysine derivative ofthe formula (5)

wherein *, P₁ and P₂ are as defined above, R₁ is alkyl group having 1 to6 carbon atoms or aralkyl group having 7 to 12 carbon atoms, and P₅ is ahydrogen atom or carboxyl-protecting group, are useful as pharmaceuticalintermediates.

For example, a compound having an S-configuration of asymmetric carbonatom is an important intermediate compound for a pharmaceutical compoundof the following formula (25), which is useful as an antihypertensiveagent having an inhibitory activity against an angiotensin convertingenzyme (ACE) and neutral endopeptidase (NEP) (Journal of MedicinalChemistry, 1999, 42, 305–311).

As a method for producing the pharmaceutical compound of the formula(25), Journal of Medicinal Chemistry, 1999, 42, 305–311 discloses amethod shown by the following scheme using(s)-2-phthalimido-6-hydroxyhexanoic acid as a starting material.

However, this method requires many steps, and there is a demand on aproduction method that permits easier and simpler industrial production.

It is also known that an optically active amino acid can be generated bydirectly applying a microorganism enzyme system to 5-substitutedhydantoin as a starting material. When a microorganism enzyme system isused, an enzyme (hydantoinase) to hydrolyze a 5-substituted hydantoincompound and produce N-carbamyl-amino acid, and an enzyme(N-carbamyl-amino-acid hydrolase) to enantio-selectively decompose theproduced N-carbamyl-amino acid into optically active amino acid arerequired.

Conventionally, there are methods for producing D-amino acid usingmicroorganisms containing these two kinds of enzymes, or substancescontaining such enzymes, such as a method comprising the use of bacteriaof the genus Pseudomonas (JP-B-56-003034), a method comprising the useof bacteria of the genus Agrobacterium (JP-A-03-019696) and the like.

As a method for producing L-amino acid, there are known a methodcomprising the use of bacteria of the genus Flavobacterium(JP-B-56-008749), a method comprising the use of bacteria of the genusBacillus (JP-A-63-24895), a method comprising the use of bacteria of thegenus Pseudomonas (JP-A-01-071476), a method comprising the use ofbacteria of the genus Arthrobacter [J. Biotechnol., Vol. 46, p. 63(1996)] and the like.

It has been also reported that optically active amino acid can beproduced by isolating a genetic DNA of hydantoinase and a genetic DNA ofN-carbamyl-amino-acid hydrolase from various bacteria and expressingthem in E. coli [Biotechnol. Prog., vol. 16, p. 564 (2000), EP515698 andthe like].

It has been detailed that the substrate specificity of thesehydantoinase and N-carbamyl-amino-acid hydrolase is rather broad, and bya combined use of these two kinds of enzymes under the limitation of thesubstrate specificity, natural or nonnatural various amino acids havingoptical activity can be produced from 5-substituted hydantoin compound[Enzyme Catalysis in Organic Synthesis, K. Drauz et al. ed., vol. 1, ch.B2.4, pp. 409–431, VCH (1995)]. Because the producible optically activeamino acid is limited due to its substrate specificity, however, anoptically active amino acid corresponding to 5-substituted hydantoincompound is not always generated. For example, Microbacteriumliquefaciens AJ3940 strain (formerly classified under Flavobacteriuimsp.) that affords L-tryptophan in a high yield from5-indolylmethylhydantoin (racemate) acts well on 5-substituted hydantoinhaving an aromatic ring, and can generate many kinds of L-enantiomer ofaromatic amino acids such as L-phenylalanine, L-tyrosine and the like.However, since it does not act at all on 5-methylhydantoin,5-sec-butylhydantoin, 5-carboxymethylhydantoin or5-carboxyethylhydantoin, and does not produce the correspondingL-alanine, L-isoleucine, L-aspartic acid or L-glutamic acid, it is knownto be specific to compounds having an aromatic ring [Agric. Biol. Chem.,vol. 51, p. 729 (1987)].

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide anindustrial method for producing optically active lysine derivativesrepresented by the aforementioned formulas (3) and (5), which are usefulas pharmaceutical intermediates.

The present inventors have conducted intensive studies in an attempt tosolve the aforementioned problems and found a completely novel reactionsequence industrially superior as a method for producing compounds ofthe aforementioned formulas (3) and (5). To be specific, it has beenfound that the aforementioned object can be achieved by the processincluding protecting an amino group and, where necessary, a carboxylgroup of optically active 2-amino-6-methyl-6-nitroheptanoic acid with aprotecting group, after which nitro group is reduced to synthesize a6,6-dimethyl lysine derivative of the formula (3), and further byreacting the 6,6-dimethyl lysine derivative with an acetic acidderivative to synthesize an optically active lysine derivative of theformula (5).

More particularly, the present invention provides the following.

-   [1] A method for producing an optically active lysine derivative of    the formula (5)    wherein-   * means an asymmetric carbon atom,-   P₁ and P₂ are each independently an amino-protecting group or    hydrogen atom where P₁ and P₂ are not hydrogen atoms at the same    time, or P₁ and P₂ in combination show an amino-protecting group,-   R₁ is alkyl group having 1 to 6 carbon atoms or aralkyl group having    7 to 12 carbon atoms, and-   P₅ is a hydrogen atom or carboxyl-protecting group    or a salt thereof, which method comprises the steps of-   (1) protecting an amino group or an amino group and a carboxyl group    of optically active 2-amino-6-methyl-6-nitroheptanoic acid of the    formula (1)    wherein * is as defined above, or a salt thereof, with a protecting    group to give an optically active amino acid derivative of the    formula (2)    wherein *, P₁ and P₂ are as defined above and P₃ is a hydrogen atom    or carboxyl-protecting group,-   (2) reducing a nitro group of the derivative of the formula (2) to    give an optically active 6,6-dimethyl lysine derivative of the    formula (3)    wherein *, P₁ and P₂ are as defined above and P₄ is a hydrogen atom    or carboxyl-protecting group, or a salt thereof, and-   (3) reacting the derivative of the formula (3) or a salt thereof    with an acetate derivative of the formula (4)    wherein Y₁ is a leaving group and R₁ is as defined above.-   [2] The method of [1], wherein the reduction is a catalytic    reduction using a transition metal catalyst and metallic sulfate.-   [3] The method of [1], wherein the reduction is a catalytic    reduction using a palladium catalyst and ferrous sulfate.-   [4] The method of [1], wherein either P₁ or P₂ is a    tert-butoxycarbonyl group and the other is a hydrogen atom, P₃, P₄    and P₅ are methyl groups and R₁ is a tert-butyl group.-   [5]A method for producing an optically active 6,6-dimethyl lysine    derivative of the aforementioned formula (3) or a salt thereof,    which method comprises protecting an amino group or an amino group    and a carboxyl group of optically active    2-amino-6-methyl-6-nitroheptanoic acid of the above-mentioned    formula (1) or a salt thereof with a protecting group to give an    optically active amino acid derivative of the above-mentioned    formula (2), and reducing a nitro group of the derivative of the    formula (2).-   [6] The method of [5], wherein the reduction is a catalytic    reduction using a transition metal catalyst and metallic sulfate.-   [7] The method of [5], wherein the reduction is a catalytic    reduction using a palladium catalyst and ferrous sulfate.-   [8] The method of [5], wherein either P₁ or P₂ is a    tert-butoxycarbonyl group and the other is a hydrogen atom and P₃    and P₄ are methyl groups.-   [9] A method for producing an optically active 6,6-dimethyl lysine    derivative of the aforementioned formula (3) or a salt thereof,    which method comprises reducing an amino acid derivative of the    above-mentioned formula (2).-   [10] The method of [9], wherein the reduction is a catalytic    reduction using a transition metal catalyst and metallic sulfate.-   [11] The method of [9], wherein the reduction is a catalytic    reduction using a palladium catalyst and ferrous sulfate.-   [12] The method of [9], wherein either P₁ or P₂ is a    tert-butoxycarbonyl group and the other is a hydrogen atom and P₃    and P₄ are methyl groups.-   [13] A method for producing an optically active lysine derivative of    the aforementioned formula (5) or a salt thereof, which method    comprises reacting an optically active 6,6-dimethyl lysine    derivative of the above-mentioned formula (3) or a salt thereof with    an acetate derivative of the above-mentioned formula (4).-   [14] The method of [13], wherein either P₁ or P₂ is a    tert-butoxycarbonyl group and the other is a hydrogen atom, P₄ and    P₅ are methyl groups and R₁ is a tert-butyl group.-   [15] The method of [1], wherein the optically active    2-amino-6-methyl-6-nitroheptanoic acid of the above-mentioned    formula (1) is produced by the following steps (a)–(c):-   (a) reacting a 4-methyl-4-nitropentane derivative of the formula (6)    wherein Y₂ is a leaving group, with 2-acylaminomalonic diester of    the formula (7)    wherein R₂ and R₃ are each independently alkyl group having 1 to 6    carbon atoms or aralkyl group having 7 to 12 carbon atoms, to give    2-acylamino-2-(4-methyl-4-nitropentyl)malonic diester of the formula    (8)    wherein R₂ and R₃ are as defined above;-   (b) subjecting 2-acylamino-2-(4-methyl-4-nitropentyl)malonic diester    of the formula (8) to hydrolysis and decarboxylation to give    2-acetylamiino-6-methyl-6-nitroheptanoic acid (racemate) of the    formula (9)    ; and-   (c) having acylase act on 2-acetylamino-6-methyl-6-nitroheptanoic    acid (racemate) of the formula (9) to give optically active    2-amino-6-methyl-6-nitroheptanoic acid of the formula (1) or a salt    thereof.-   [16] The method of [1], wherein the optically active    2-amino-6-methyl-6-nitroheptanoic acid of the above-mentioned    formula (1) is produced by the following steps (d)–(g):-   (d) reducing 5-(4-methyl-4-nitropentylidene)hydantoin of the formula    (10)    to give 5-(4-methyl-4-nitropentyl)hydantoin of the formula (11)-   (e) hydrolyzing 5-(4-methyl-4-nitropentyl)hydantoin of the    formula (11) to give 2-amino-6-methyl-6-nitroheptanoic acid    (racemate) of the formula (12)-   (f) acylating an amino group of 2-amino-6-methyl-6-nitroheptanoic    acid of the formula (12) to give    2-acylamino-6-methyl-6-nitroheptanoic acid (racemate) of the formula    (13)    wherein R₄ is methyl group or phenyl group; and-   (g) having acylase act on 2-acylamino-6-methyl-6-nitroheptanoic acid    (racemate) of the formula (13) to give optically active    2-amino-6-methyl-6-nitroheptanoic acid of the formula (1) or a salt    thereof.-   [17] A compound of any of the formulas (14)–(18), a salt thereof, an    optically active substance thereof or a racemate thereof:    wherein P₁ and P₂ are each independently an amino-protecting group    or hydrogen atom where P₁ and P₂ are not hydrogen atoms at the same    time, or P₁ and P₂ in combination show an amino-protecting group    except phthaloyl group, P_(3a) is a carboxyl-protecting group and P₄    is a hydrogen atom or a carboxyl-protecting group.-   [18] A compound of any of the formulas (19)–(23):    or a salt thereof.-   [19] A compound of any of the formulas (6), (8), (10), (11) and (24)    or a salt thereof:    wherein Y₂ is a leaving group except chlorine atom, R₄ is methyl    group or phenyl group, and R₂ and R₃ are each independently alkyl    group having 1 to 6 carbon atoms or aralkyl group having 7 to 12    carbon atoms, and wherein the compounds of the formulas (8) and (24)    comprise their optically active substances and racemates.-   [20] The method of [1], wherein the optically active    2-amino-6-methyl-6-nitroheptanoic acid of the above-mentioned    formula (1) is produced via the following step (h):-   (h) enantio-selectively hydrolyzing    5-(4-methyl-4-nitropentyl)hydantoin of the formula (11)    using at least one member selected from the group consisting of a    microorganism, a treated product of a microorganism, hydantoinase    and N-carbamyl-amino-acid hydrolase to give optically active    2-amino-6-methyl-6-nitroheptanoic acid of the formula (1) or a salt    thereof.-   [21] The method of [20], wherein said microorganism, and the origin    of said treated product of a microorganism, said hydantoinase and    said N-carbamyl-amino-acid hydrolase are bacteria of the genus    Agrobacterium, the genus Bacillus or the genus Microbacterium.-   [22] The method of claim [20], wherein said microorganism, and the    origin of said treated product of a microorganism, said hydantoinase    and said N-carbamyl-amino-acid hydrolase are bacteria of the genus    Agrobacterium, and the optically active    2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof is    D-2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof.-   [23] The method of claim [20], wherein said microorganism, and the    origin of said treated product of a microorganism, said hydantoinase    and said N-carbamyl-amino-acid hydrolase are bacteria of the genus    Bacillus or the genus Microbacterium, and said optically active    2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof is    L-2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof.

DETAILED DESCRIPTION OF THE INVENTION

In the formulas of the present invention, P₁ and P₂ are eachindependently an amino-protecting group or hydrogen atom, where P₁ andP₂ are not hydrogen atoms at the same time (that is, when the aminogroup does not have a protecting group). Alternatively, P₁ and P₂ incombination form an amino-protecting group. The amino-protecting groupis not particularly limited, and may be, for example, the protectinggroup and the like disclosed in Protecting Groups in Organic Chemistry2nd edition (John Wiley & Sons, Inc. 1991). A typical protecting groupis exemplified by benzyloxycarbonyl group (Z group),9-fluorenylmethoxycarbonyl group (Fmoc group), tert-butoxycarbonyl group(Boc group), methoxycarbonyl group (Moc group) and the like. Theprotecting group when P₁ and P₂ in combination form an amino-protectinggroup is exemplified by phthaloyl group and the like.

According to the present invention, one of P₁ and P₂ is particularlypreferably tert-butoxycarbonyl group, and the other is hydrogen atom.

In the formulas of the present invention, P₃, P₄ and P₅ are eachhydrogen atom or carboxyl-protecting group. The carboxyl-protectinggroup is not particularly limited, and may be, for example, alkyl grouphaving 1 to 6 carbon atoms, aryl group having 6 to 10 carbon atoms andthe like. Specific examples thereof include methyl group, ethyl group,n-propyl group, isopropyl group, n-butyl group, isobutyl group,sec-butyl group, t-butyl group, phenyl group and the like. Thecarboxyl-protecting group shown by P₃, P₄ and P₅ in the presentinvention is particularly preferably methyl group.

In the formulas of the present invention, P_(3a) is acarboxyl-protecting group which is the same as the aforementionedcarboxyl-protecting group at P₃.

In the formulas of the present invention, R₁ is alkyl group having 1 to6 carbon atoms or aralkyl group having 7 to 12 carbon atoms. Examples ofthe alkyl group having 1 to 6 carbon atoms include methyl group, ethylgroup, n-propyl group, isopropyl group, n-butyl group, isobutyl group,sec-butyl group, t-butyl group and the like. Examples of the aralkylgroup having 7 to 12 carbon atoms include benzyl group and the like. AsR₁, tert-butyl group is particularly preferable.

In the formulas of the present invention, R₂ and R₃ are eachindependently alkyl group having 1 to 6 carbon atoms or aralkyl grouphaving 7 to 12 carbon atoms. Examples of alkyl group having 1 to 6carbon atoms include methyl group, ethyl group, n-propyl group,isopropyl group, n-butyl group, isobutyl group, sec-butyl group, t-butylgroup and the like. Examples of aralkyl group having 7 to 12 carbonatoms include benzyl group and the like. As R₂ and R₃, alkyl grouphaving 1 to 6 carbon atoms is preferable, alkyl group having 1 to 4carbon atoms is more preferable, and ethyl group is particularlypreferable. R₂ and R₃ are preferably the same.

In the present invention, Y₁ in the formula (4) and Y₂ in the formula(6) are each a leaving group. The leaving group means an atom or atomicgroup that is released from a reaction substrate by a substitutionreaction, elimination reaction and the like, and is exemplified bychlorine, bromine, iodine, p-toluenesulfonyloxy group, mesyloxy group,trifluoromethanesulfonyloxy group, alkylcarbonate group, phenylcarbonategroup, or saturated or unsaturated acyloxy group having 1 to 8 carbonatoms and the like.

A preferable leaving group is bromine atom or iodine atom.

A typical compound preferably produced by the production method of thepresent invention is a compound of the formula (3), such asNα-t-butoxycarbonyl-6,6-dimethyl-L-lysine methyl ester of the followingformula (23), and a compound of the formula (5), such asNα-t-butoxycarbonyl-Nε-t-butoxycarbonylmethyl-6,6-dimethyl-L-lysinemethyl ester of the formula (26).

A method for producing the amino acid derivative of the aforementionedformula (1), which is the starting material in the present invention, isexplained in the following.

The amino acid derivative of the formula (1) can be synthesized by threeroutes (I), (II) and (III) as shown in the following reaction scheme.

The route (I) is explained in the following.

(i) A catalytic amount of potassium fluoride is added to 2-nitropropaneand methyl acrylate, and the mixture is refluxed under heating in asuitable solvent such as ethanol and the like. The reaction mixture isconcentrated (or evaporated) and extracted to give methyl4-methyl-4-nitropentanoate (Bulletin of the Chemical Society of Japan1966, 39 (11), 2549–2551). The obtained methyl4-methyl-4-nitropentanoate can be isolated and/or purified by aconventional method, such as distillation, chromatography and the like,but generally, can be used in the next step without isolation and/orpurification.

(ii) Methyl 4-methyl-4-nitropentanoate obtained above is reacted with atleast 2 equivalents of a reducing agent, such as sodium borohydride andthe like, in a suitable solvent, such as ethanol and the like, bystirring the mixture at room temperature—a refluxing temperature,preferably 40° C.–70° C., for about 1–24 h, preferably about 2–5 h. Thereaction mixture is concentrated (or evaporated) and extracted to give4-methyl-4-nitropentanol. The obtained 4-methyl-4-nitropentanol can beisolated and/or purified by a conventional method, such as distillation,chromatography and the like, but generally, can be used in the next stepwithout isolation and/or purification.

(iii) By substituting the hydroxyl group of 4-methyl-4-nitropentanol fora leaving group, a 4-methyl-4-nitropentane derivative of theaforementioned formula (6) is obtained by a method known to those ofordinary skill in the art. For example, substitution for a preferableleaving group, such as halogen atom (e.g., bromine atom, iodine atometc.) can be performed according to the following method.

For example, about 1–3 equivalents, preferably about 1.2–2 equivalents,of tertiary amine, such as triethylamine and the like, and about 1–3equivalents, preferably about 1.1–2 equivalents, of sulfonyl halide ofthe formula (27) (preferably methanesulfonyl chloride) are added to4-methyl-4-nitropentanol in a suitable solvent, such as methylenechloride and the like, and the mixture is stirred at about roomtemperature—0° C. for 30 min–2 h, after which the reaction mixture isextracted to give 4-methyl-4-nitropentanol sulfonate of the formula(28). The obtained 4-methyl-4-nitropentanol sulfonate is generallyisolated and/or purified before use by a conventional method such ascrystallization, chromatography and the like.

Then, 4-methyl-4-nitropentanol sulfonate of the formula (28) is reactedwith alkali metal halide to give 4-methyl-4-nitropentyl halide of theformula (29). Examples of preferable alkali metal halide include sodiumiodide and sodium bromide. For example, about 1–10 equivalents,preferably about 4–5 equivalents, of alkali metal halide is added to4-methyl-4-nitropentanol sulfonate of the formula (28) in a suitablesolvent, such as acetone and the like, and the mixture is stirred atabout 0° C.–30° C., preferably about 15° C.–25° C. for about 6–24 h,preferably about 10–12 h, after which the reaction mixture isconcentrated (or evaporated) and extracted to give4-methyl-4-nitropentyl halide of the formula (29). The obtained4-methyl-4-nitropentyl halide can be isolated and/or purified by aconventional method, such as chromatography and the like, but generally,can be used in the next step without isolation and/or purification.

wherein R₅ is alkyl group having 1 to 3 carbon atoms or phenyl groupoptionally having a substituent (preferably alkyl group having 1 to 3carbon atoms) and X₁ and X₂ are halogen atoms.

(iv) 4-Methyl-4-nitropentane derivative of the formula (6) (preferably4-methyl-4-nitropentyl halide of the formula (29)) and2-acetylaminomalonic diester of the formula (7) are reacted to give2-acetylamino-2-(4-methyl-4-nitropentyl)malonic diester (racemate) ofthe aforementioned formula (8).

As 2-acetylaminomalonic diester of the formula (7), 2-acetamidomalonicacid diethyl ester wherein R₂ and R₃ are ethyl groups is particularlypreferably used. For example, sodium ethoxide and 2-acetamidomalonicdiester are dissolved in a suitable solvent, such as ethanol and thelike, and 4-methyl-4-nitropentyl derivative is added. The mixture isreacted at about 50° C.—refluxing temperature for about 3–24 h. Thereaction mixture is concentrated (or evaporated) and extracted to give2-acetylamino-2-(4-methyl-4-nitropentyl)malonic diester (racemate) ofthe formula (8) (Tetrahedron, 1985, 41 (22), 5307–5311). The obtained2-acetylamino-2-(4-methyl-4-nitropentyl)malonic diester (racemate) canbe isolated and/or purified by a conventional method, such aschromatography and the like, but generally, can be used in the next stepwithout isolation and/or purification.

(v) Alkali, such as potassium hydroxide and the like, is added to2-acetylamino-2-(4-methyl-4-nitropentyl)malonic diester of the formula(8) in a mixed solvent of a water soluble solvent, such as ethanol andthe like, and water, and the mixture is refluxed under heating to give2-acetylamino-2-(4-methyl-4-nitropentyl)malonic acid. Thereto is addedan acid, such as hydrochloric acid, sulfuric acid and the like, and themixture is subjected to decarboxylation. The reaction mixture isconcentrated by crystallization, or extracted to give2-acetylamino-6-methyl-6-nitroheptanoic acid (racemate) of the formula(9) (Journal of Medicinal Chemistry, 1999, 42, 305–311). The obtained2-acetylamino-6-methyl-6-nitroheptanoic acid (racemate) is generallyisolated and/or purified before use by a conventional method, such ascrystallization, chromatography and the like.

(vi) Acylase is made to act on 2-acetylamino-6-methyl-6-nitroheptanoicacid (racemate) of the formula (9) to give optically active2-amino-6-methyl-6-nitroheptanoic acid of the formula (1). Acylase isnot particularly limited, and conventionally available acylase (e.g.,acylase manufactured by Amano Enzyme Inc.) and the like can be used. Forexample, L-acylase is added to an aqueous solvent adjusted to pH 5–10,preferably 6–9, containing 2-acetylamino-6-methyl-6-nitroheptanoic acid(racemate), and the mixture is preferably stirred at 30° C.–40° C. forabout 3 h–2 days. The pH is adjusted to about 1–3 to precipitateunreacted D-enantiomer of the formula (9). After separation byfiltration, the mother liquor is concentrated to dryness to giveL-enantiomer of 2-amino-6-methyl-6-nitroheptanoic acid. When D-acylaseis used here, D-enantiomer of 2-amino-6-methyl-6-nitroheptanoic acid canbe obtained (Journal of Medicinal Chemistry, 1999, 42, 305–311).

As a different method, 2-acetylamino-6-methyl-6-nitroheptanoic acid(racemate) is treated with D-acylase to deacetylate D-enantiomer andunreacted L-enantiomer is crystallized under acidic conditions. TheL-enantiomer of 2-amino-6-methyl-6-nitroheptanoic acid can be alsoobtained by separation of crystals, refluxing under heating in an acidicaqueous solution, deacetylation and concentration to dryness.

The obtained optically active 2-amino-6-methyl-6-nitroheptanoic acid canbe isolated and/or purified by a conventional method, such ascrystallization, chromatography and the like, but generally, can be usedin the next step without isolation and/or purification.

The route (II) is explained in the following.

(vii) The method for producing 4-methyl-4-nitropentanal is exemplifiedby the following two methods.

(vii-1) Acrylonitrile and 2-nitropropane are reacted in the presence ofhexadecyltrimethyl ammonium chloride in an aqueous alkali solvent suchas aqueous sodium hydroxide solution and the like. Then, the reactionmixture is extracted to give 4-methyl-4-nitrovaleronitrile (EuropeanJournal of Organic Chemistry (1998), (2), 355–357). The obtained4-methyl-4-nitrovaleronitrile can be isolated and/or purified by aconventional method, such as crystallization, chromatography and thelike, but generally, can be used in the next step without isolationand/or purification.

4-Methyl-4-nitrovaleronitrile is reduced in a suitable solvent, such asmethylene chloride and the like, with a reducing agent, such asdiisobutylaluminum hydride and the like, at about 0° C. to −78° C.,preferably from about −40° C. to −60° C., and treated with an acid, suchas hydrochloric acid, sulfuric acid and the like, after which extractedto give 4-methyl-4-nitropentanal. The obtained 4-methyl-4-nitropentanalcan be isolated and/or purified by a conventional method, such asdistillation, chromatography and the like, but generally, can be used inthe next step without isolation and/or purification.

(vii-2) It is also possible to obtain 4-methyl-4-nitropentanal by adding2-nitropropane and acrolein to a liquid in which methanol and metallicsodium have been dissolved (see JP-A-01-305056). The obtained4-methyl-4-nitropentanal can be isolated and/or purified before use by aconventional method, such as distillation, chromatography and the like.

(viii) Then, 4-methyl-4-nitropentanal and hydantoin are reacted to give5-(4-methyl-4-nitropentylidene)hydantoin (see JP-A-11-140076).

For example, 4-methyl-4-nitropentanal and hydantoin are refluxed underheating in a mixed solvent of water soluble solvent, such asacetonitrile, isopropyl alcohol and the like, and-water in the presenceof a base, such as sodium carbonate, potassium carbonate and the like,preferably for about 3–5 days. Extraction thereafter gives5-(4-methyl-4-nitropentylidene)hydantoin.

In this reaction, 5-(1-hydroxy-4-methyl-4-nitropentyl)hydantoin isgenerated as an intermediate product which is ultimately converted tothe objective 5-(4-methyl-4-nitropentylidene)hydantoin.

It is also possible to obtain the objective5-(4-methyl-4-nitropentylidene)hydantoin by isolating5-(1-hydroxy-4-methyl-4-nitropentyl)hydantoin, protecting a hydroxylgroup with methanesulfonyl chloride and the like to give5-(1-methanesulfonyloxy-4-methyl-4-nitropentyl)hydantoin and the like,which is then treated with a base such as1,8-diazabicyclo[5.4.0]undec-7-ene(DBU) and the like.

The obtained 5-(4-methyl-4-nitropentylidene)hydantoin can be isolatedand/or purified by a conventional method, such as crystallization,chromatography and the like, but generally, can be used in the next stepwithout isolation and/or purification.

(viv) 5-(4-Methyl-4-nitropentylidene)hydantoin is reduced with atransition metal catalyst (such as palladium) and the like to give5-(4-methyl-4-nitropentyl)hydantoin. For example,5-(4-methyl-4-nitropentylidene)hydantoin is reacted with hydrogen gasupon addition of palladium-carbon (generally 0.1–5 mol %, preferably0.5–2 mol %) in a mixed solvent of a water soluble solvent, such asmethanol, ethanol and the like, and water for 1–12 h, preferably 2–5 h.The reaction solvent is concentrated to dryness to give5-(4-methyl-4-nitropentyl)hydantoin. The obtained5-(4-methyl-4-nitropentyl)hydantoin can be isolated and/or purified by aconventional method, such as chromatography and the like, but generally,can be used in the next step without isolation and/or purification.

(x) 5-(4-Methyl-4-nitropentyl)hydantoin is hydrolyzed with alkali togive 2-amino-6-methyl-6-nitroheptanoic acid (see JP-A-11-140076). Forexample, 5-(4-methyl-4-nitropentyl)hydantoin is hydrolyzed in an aqueoussolution preferably in the presence of 1–3 equivalents of alkali, suchas sodium hydroxide, potassium hydroxide, sodium carbonate, potassiumcarbonate, calcium hydroxide, barium hydroxide and the like, relative to5-(4-methyl-4-nitropentyl)hydantoin generally at 20–200° C., preferably100–150° C. The reaction mixture is concentrated to dryness to give2-amino-6-methyl-6-nitroheptanoic acid. The obtained2-amino-6-methyl-6-nitroheptanoic acid can be isolated and/or purifiedby a conventional method, such as crystallization, chromatography andthe like, but generally, can be used in the next step without isolationand/or purification.

(xi) The amino group of 2-amino-6-methyl-6-nitroheptanoic acid isacylated to give 2-acylamino-6-methyl-6-nitroheptanoic acid (racemate)of the formula (13) (see JP-A-11-140076). For example,2-amino-6-methyl-6-nitroheptanoic acid is acylated in an aqueoussolution adjusted to a pH higher than pH 7, preferably pH 8–11,preferably using 1–2 equivalents of acyl halide, such as acetylchloride, benzoyl chloride and the like, or acid anhydride, such asacetic anhydride, benzoic anhydride and the like, generally at 0–80° C.,preferably 0–30° C., to give 2-acylamino-6-methyl-6-nitroheptanoic acid(racemate). The obtained 2-acylamino-6-methyl-6-nitroheptanoic acid isisolated and/or purified before use by a conventional method such ascrystallization, chromatography and the like.

(xii) Acylase is made to act on 2-acylamino-6-methyl-6-nitroheptanoicacid obtained as mentioned above according to the aforementioned methodto give optically active 2-amino-6-methyl-6-nitroheptanoic acid of theformula (1).

The optically active 2-amino-6-methyl-6-nitroheptanoic acid of theformula (1) can be also produced by the following route (III). That is,5-(4-methyl-4-nitropentyl)hydantoin of the formula (11) isenantio-selectively hydrolyzed using at least one member selected fromthe group consisting of a microorganism, a treated product thereof,hydantoinase and N-carbamyl-amino-acid hydrolase to give opticallyactive 2-amino-6-methyl-6-nitroheptanoic acid of the formula (1) or asalt thereof.

The route (III) is explained in the following.

The optically active 2-amino-6-methyl-6-nitroheptanoic acid of theformula (1) can be also produced by enantio-selectively hydrolyzing5-(4-methyl-4-nitropentyl)hydantoin of the formula (11) using at leastone member selected from the group consisting of a microorganism, atreated product thereof, hydantoinase and N-carbamyl-amino-acidhydrolase.

The microorganism, and the origin of the treated product of amicroorganism, hydantoinase and N-carbamyl-amino-acid hydrolase to beused here are preferably bacteria of the genus Agrobacterium, the genusBacillus or the genus Microbacterium.

The microorganism, and the origin of the treated product of amicroorganism, hydantoinase and N-carbamyl-amino-acid hydrolase to beused here are preferably bacteria of the genus Agrobacterium, and theoptically active 2-amino-6-methyl-6-nitroheptanoic acid or a saltthereof is preferably D-2-amino-6-methyl-6-nitroheptanoic acid or a saltthereof.

Further, the microorganism, and the origin of the treated product of amicroorganism, hydantoinase and N-carbamyl-amino-acid hydrolase to beused here are preferably bacteria of the genus Microbacterium or thegenus Bacillus, and the optically active2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof is preferablyL-2-amino-6-methyl-6-nitroheptanoic acid or a salt thereof.

5-(4-Methyl-4-nitropentyl)hydantoin of the formula (11) can be preparedby a method of the route (II). The 5-(4-methyl-4-nitropentyl)hydantointo be prepared may be purified or isolated by a conventional method orcontained in a reaction mixture. 5-(4-Methyl-4-nitropentyl)hydantoin maybe a mixture of a D-enantiomer and an L-enantiomer, or either of them.In the case of a mixture of a D-enantiomer and an L-enantiomer, themixing ratio is optional.

The microorganism, a treated product thereof, hydantoinase andN-carbamyl-amino-acid hydrolase to be used here may be any as long asthey can hydrolyze 5-(4-methyl-4-nitropentyl)hydantoinenantio-selectively.

The enantio-selective hydrolysis is a reaction capable of affordingoptically active 2-amino-6-methyl-6-nitroheptanoic acid of the formula(1) or a salt thereof by hydrolysis of5-(4-methyl-4-nitropentyl)hydantoin of the formula (11), wherein eitherD-enantiomer or L-enantiomer of 2-amino-6-methyl-6-nitroheptanoic acidor a salt thereof is generated.

The microorganism, a treated product thereof and hydantoinase includethose that can act on 5-(4-methyl-4-nitropentyl)hydantoin, andenantio-selectively hydrolyze same, by which to generate opticallyactive N-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid, which isspecifically N-carbamyl-L-2-amino-6-methyl-6-nitroheptanoic acid orN-carbamyl-D-2-amino-6-methyl-6-nitroheptanoic acid. This is because,even when the microorganism and the treated product thereof do not havean activity to enantio-selectively convertN-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid to2-amino-6-methyl-6-nitroheptanoic acid, or even when hydantoinase alonecatalyzes an enantio-selective reaction, optically active2-amino-6-methyl-6-nitroheptanoic acid can be produced in a high yieldwhile maintaining optical activity by continuously applying an enzymatichydrolysis treatment using N-carbamyl-amino-acid hydrolase or asubstance containing this enzyme, or by applying a chemical hydrolysistreatment by nitrous acid.

The microorganism, a treated product thereof and N-carbamyl-amino-acidhydrolase include those that can act onN-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid, andenantio-selectively hydrolyze same, by which to produce optically active2-amino-6-methyl-6-nitroheptanoic acid. This is because, even when themicroorganism and the treated product thereof do not have an activity toenantio-selectively convert 5-(4-methyl-4-nitropentyl) hydantoin toN-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid, or even whenN-carbamyl-amino-acid hydrolase alone catalyzes an enantio-selectivereaction, optically active 2-amino-6-methyl-6-nitroheptanoic acid can beproduced in a high yield by previously applying an enzymatic hydrolysistreatment using hydantoinase or a substance containing this enzyme, orby applying a chemical hydrolysis treatment.

Therefore, any microorganism or any treated product thereof can be usedin the present invention as long as it contains hydantoinase that canact on 5-(4-methyl-4-nitropentyl)hydantoin and perform enantio-selectivehydrolysis, thereby to generate optically activeN-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid. Furthermore, anymicroorganism or any treated product thereof can be used even if itcontains hydantoinase without enantio-selectivity, only if it containsenantio-selective N-carbamyl-amino-acid hydrolase that canenantio-selectively hydrolyze the generatedN-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid, thereby to produceoptically active 2-amino-6-methyl-6-nitroheptanoic acid.

The microorganism as referred to in the present invention can becultured by any culture method, such as liquid culture, solid cultureand the like, as long as it maintains the required ability. Both theculture broth itself and viable bacterial cells harvested from theculture broth can be used. The microorganism of the present inventionmay be a strain newly isolated from nature, such as soil and plant, ormay be a strain artificially bred by mutagen treatment, recombinant DNAtechnology and the like.

For culture of microorganism in the present invention, a mediumgenerally employed in this field is used, such as a medium containingcarbon source, nitrogen source, mineral, trace metal salts, vitamingroup and the like. Depending on the kind of microorganism and cultureconditions, a 5-substituted hydantoin compound such as5-indolylmethylhydantoin or 5-isopropylhydantoin may be added to themedium in a proportion of about 0.05–1.0 g/dl to promote the activity toproduce optically active 2-amino-6-methyl-6-nitroheptanoic acid. Toincrease permeability of 5-(4-methyl-4-nitropentyl)hydantoin (substrate)into a bacterial cell, a surfactant, such as Triton X and Tween, and/oran organic solvent, such as toluene and xylene, can be used. Referringto the specific substances to be used as the components of theabove-mentioned medium, for example, the carbon source is not subject toany particular limitation as long as the microorganism to be used canconsume, and glucose, sucrose, fructose, glycerol, maltose, acetic acidand a mixture of these can be used. As the nitrogen source, ammoniumsulfate, ammonium chloride, urea, yeast extract, meat extract, cornsteep liquor, casein hydrolysate and a mixture of these can be used. Aconcrete medium composition is, for example, a medium containing glucose0.5%, ammonium sulfate 0.5%, powder yeast extract 1.0%, peptone 1.0%,KH₂PO₄ 0.1%, K₂HPO₄ 0.3%, MgSO₄.7H₂O 0.05%, FeSO₄.7H₂O 0.001% andMnSO₄.5H₂O 0.001% (pH 7.0) and the like.

The culture temperature is generally within the range where themicroorganism to be used can grow, which is 20–45° C., preferably 25–37°C. The pH of the medium is adjusted to 3–11, preferably 4–8. Theaeration condition is aerobic or anaerobic and set suitably for thegrowth of the microorganism to be used, with preference given to aerobiccondition. The culture time is not particularly limited as long asoptically active 2-amino-6-methyl-6-nitroheptanoic acid is generatedefficiently. It is generally about 12–144 h, preferably about 24–96 h.

The microorganism to be used in the present invention is preferably amicroorganism belonging to the genus Agrobacterium for producing(R)-2-amino-6-methyl-6-nitroheptanoic acid, and a microorganismbelonging to the genus Microbacterium or the genus Bacillus forproducing (S)-2-amino-6-methyl-6-nitroheptanoic acid.

Specific example of the microorganism used for producing, for example,(R)-2-amino-6-methyl-6-nitroheptanoic acid is Agrobacterium sp. AJ11220strain (formerly classified under Pseudomonas hydantoinophilum). TheAgrobacterium sp. AJ11220 strain was deposited at the then NationalInstitute of Bioscience and Human-Technology, Agency of IndustrialScience and Technology, Ministry of International Trade and Industry(now International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology, an IndependentAdministrative Institution under Ministry of Economy, Trade andIndustry) on Dec. 20, 1977 under the accession number of FERM-P4347, andthen transferred to an international deposition under the BudapestTreaty on Jun. 27, 2001, and received a accession number of FERMBP-7645. For the production of (S)-2-amino-6-methyl-6-nitroheptanoicacid, moreover, Microbacterium liquefaciens AJ3940 (formerly classifiedunder Aureobacterium liquefaciens still formerly classified underFlavobacterium sp.), Bacillus sp. AJ12299 can be mentioned.Microbacterium liquefaciens AJ3940 strain was deposited on Jun. 27,1975, and Bacillus sp. AJ12299 strain was deposited on Jul. 5, 1986, atthe then National Institute of Bioscience and Human-Technology, Agencyof Industrial Science and Technology, Ministry of International Tradeand Industry (now International Patent organism Depositary, NationalInstitute of Advanced Industrial Science and Technology, an IndependentAdministrative Institution under Ministry of Economy, Trade andIndustry) under the accession number of FERM-P3135 and FERM-P8837,respectively, and then transferred to an international deposition underthe Budapest Treaty both on Jun. 27, 2001, and received a accessionnumber of FERM BP-7644 and FERM BP-7646, respectively.

The treated product of microorganism as referred to in the presentinvention is obtained by subjecting the microorganism to be used in thepresent invention to a physical treatment using, for example,ultrasonication, glass beads, French press, lyophillization and thelike; enzymatic treatment using lytic enzyme etc.; chemical treatmentusing an organic solvent, surfactant etc.; and the like. The treatedproduct of microorganism in the present invention may be a culture brothof a microorganism or viable bacterial cells, that underwent suchtreatments, as long as it has the required ability. Furthermore, a crudefractionation enzyme or purification enzyme prepared by a conventionalmethod (liquid chromatography, ammonium sulfate fractionation etc.) froma substance that underwent such treatments may be used as the treatedproduct of microorganism in the present invention, as long as it has therequired ability.

The origin of the treated product of microorganism is preferably, forexample, a microorganism belonging to the genus Agrobacterium forproducing (R)-2-amino-6-methyl-6-nitroheptanoic acid, and amicroorganism belonging to the genus Microbacterium or the genusBacillus for producing (S)-2-amino-6-methyl-6-nitroheptanoic acid.

The origin of the treated product of microorganism means themicroorganism before the treatment. However, the origin of the treatedproduct of microorganism obtained by treating a transformant prepared byisolating a gene of hydantoinase from a microorganism having ahydantoinase activity, or isolating a gene of N-carbamyl-amino-acidhydrolase from a microorganism having an N-carbamyl-amino-acid hydrolaseactivity, and transforming a microorganism with the gene is themicroorganism from which the gene was isolated.

A hydantoinase that hydrolyzes a hydantoin compound enantio-selectivelycan be obtained as follows. For example, bacteria having D-hydantoinasethat produces N-carbamyl-D-amino acid is known to be the bacteria of thegenus Bacillus having a heat resistant enzyme. Thus, a fractioncontaining hydantoinase or hydantoinase can be prepared from, forexample, Bacillus stearothermophilus ATCC31195 and the like [Appl.Microbiol. Biotechnol., Vol. 43, p. 270 (1995)]. The ATCC31195 strain isavailable from the American Type Culture Collection (address: 12301Parklawn Drive, Rockville, Md. 20852, United States of America). TheL-hydantoinase known to specifically act on an L-enantiomer of hydantoincompound is known to exist in, for example, the above-mentioned Bacillussp. AJ12299 strain (JP-A-63-24894).

A hydantoinase without enantio-selectivity is known to exist inMicrobacterium liquefaciens AJ3912 (formerly classified underAureobacterium liquefaciens still formerly classified underFlavobacterium sp.) and the above-mentioned Microbacterium liquefaciensAJ3940 (JP-B-56-008749), as well as, for example, Arthrobacter aurescens[J. Biotechnol., vol. 61, p. 1, (1998)]. The AJ3912 strain was depositedat the then National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology, Ministry of InternationalTrade and Industry (now International Patent organism Depositary,National Institute of Advanced Industrial Science and Technology, anIndependent Administrative Institution under Ministry of Economy, Tradeand Industry) on Jun. 27, 1975 under the deposit No. of FERM-P3133, andthen transferred to an international deposition under the BudapestTreaty on Jun. 27, 2001, and received a accession number of FERMBP-7643.

The N-carbamyl-amino-acid hydrolase that hydrolyzes N-carbamyl-aminoacid in a D-enantiomer-selective manner is known to exist in theabove-mentioned Agrobacterium sp. AJ11220 strain (JP-B-56-003034). TheN-carbamyl-amino-acid hydrolase that hydrolyzes N-carbamyl-amino acid inan L-enantiomer-selective manner is known to exist in theabove-mentioned Microbacterium liquefaciens AJ3912 (JP-B-56-008749) andBacillus sp. AJ12299.

The preferable origin of hydantoinase and N-carbamyl-amino-acidhydrolase is, for example, a microorganism belonging to the genusAgrobacterium for producing (R)-2-amino-6-methyl-6-nitroheptanoic acid(D-enantiomer), and a microorganism belonging to the genusMicrobacterium or the genus Bacillus for producing(S)-2-amino-6-methyl-6-nitroheptanoic acid (L-enantiomer).

By the origin of hydantoinase is meant the resources from which thehydantoinase is obtained. The origin of hydantoinase obtained by lysisof cells of a microorganism having a hydantoinase activity is thismicroorganism. However, the origin of hydantoinase obtained from atransformant obtained by isolating a hydantoinase gene from amicroorganism having a hydantoinase activity and transforming amicroorganism with the gene is the microorganism from which the gene wasisolated.

By the origin of N-carbamyl-amino-acid hydrolase is meant the resourcesfrom which the N-carbamyl-amino-acid hydrolase is obtained. The originof N-carbamyl-amino-acid hydrolase obtained by lysis of cells of amicroorganism having an N-carbamyl-amino-acid hydrolase activity is thismicroorganism. However, the origin of N-carbamyl-amino-acid hydrolaseobtained from a transformant obtained by isolating anN-carbamyl-amino-acid hydrolase gene from a microorganism having anN-carbamyl-amino-acid hydrolase activity and transforming amicroorganism with the gene is the microorganism from which the gene wasisolated.

The 5-(4-methyl-4-nitropentyl)hydantoin to be the substrate is added toa reaction system containing bacterial cell separated after culture ofthe microorganism, a treated product thereof, hydantoinase orN-carbamyl-amino-acid hydrolase, all at once or intermittently orcontinuously within the concentration range where the production ofoptically active 2-amino-6-methyl-6-nitroheptanoic acid is not limited.The method for addition may be direct addition into the culture of themicroorganism. An organic solvent or a surfactant may be added to thereaction system together with the substrate for the purpose ofincreasing the solubility or promoting dispersion. In addition, mediumcomponents, such as carbon source and nitrogen source, may be added tothe reaction system together with the substrate for the purpose ofcontinuing or promoting the metabolism of the microorganism.

When a microorganism, a treated product thereof, hydantoinase orN-carbamyl-amino-acid hydrolase is subjected to a reaction, it may beincluded in carrageenan gel or polyacrylamide, or immobilized on amembrane of polyether sulfone, regenerated cellulose and the like.

The reaction system of the present invention is obtained by adjusting areaction mixture containing a hydantoin compound and a microorganism, atreated product thereof, hydantoinase or N-carbamyl-amino-acid hydrolaseat a suitable temperature of 25–40° C. and standing or stirring themixture for 8 h–5 days while maintaining pH 5–9.

The amount of (R)- or (S)-2-amino-6-methyl-6-nitroheptanoic acid in theculture broth or the reaction mixture can be measured quickly accordingto a known method. For example, high performance liquid chromatographyusing an optical resolution column such as “CROWNPAK CR(+)” manufacturedby DAICEL CHEMICAL INDUSTRIES, LTD. can be applied. In this way, (R)- or(S)-2-amino-6-methyl-6-nitroheptanoic acid accumulated in the culturebroth or the reaction mixture can be harvested from the culture broth orthe reaction mixture by a conventional method and used. Harvesting fromthe culture broth or the reaction mixture can be done according to amethod generally used in the pertinent field for this purpose, such asfiltration, centrifugation, concentration in vacuo, ion-exchange oradsorption chromatography, crystallization and the like, which may becombined as appropriate.

The production method of the present invention shown in the following isnow explained.

A method wherein the amino group or amino group and carboxyl group ofthe optically active 2-amino-6-methyl-6-nitroheptanoic acid of theformula (1) are protected by a protecting group to produce an opticallyactive amino acid derivative of the formula (2) is explained first.

The protection by a protecting group may be first applied to eitheramino group or carboxyl group. The carboxyl group is not necessarilyprotected during reduction, and the protection may not be applied up tothe compound of the formula (5), depending on the purpose. Whenprotected, the protection before reduction is preferable, but protectionin an optional process thereafter is also acceptable. The claims of thepresent invention encompass such mode of the process.

The amino group can be protected by applying an amino group-protectingreagent, such as alkoxycarbonylating reagent, acylating reagent,sulfonylating reagent and the like, where necessary, in the presence ofa base.

For example, a compound of the formula (1) or the formula (30) isdissolved in advance in a suitable solvent by adding, where necessary, asuitable base and an amino group-protecting reagent, such asalkoxycarbonylating reagent, acylating reagent, sulfonylating reagentand the like.

The amino group-protecting reagent is not particularly limited, and anycompound containing a functional group, such as alkoxycarbonyl group,acyl group, sulfonyl group and the like, can be used for introducing anysubstituent, not to mention reagents generally used for peptidesynthesis.

Examples of the amino group-protecting reagent includealkoxycarbonylating reagent such as methoxycarbonyl chloride,ethoxycarbonyl chloride, t-butoxycarbonyl chloride, benzyloxycarbonylchloride, di-t-butyldicarbonate, tetrahydrofuran-3-yloxycarbonylchloride and the like; acylating reagent such as acetic anhydride,acetyl chloride, benzoyl chloride, trifluoroacetic anhydride and thelike; sulfonylating reagent such as methanesulfonyl chloride,trifluoromethanesulfonyl chloride, benzenesulfonyl chloride,p-toluenesulfonyl chloride and the like; and the like.

Particularly, protection with a t-butoxycarbonyl group is preferable. Inthis case, a preferable protecting reagent is di-t-butyldicarbonate.

The amino group-protecting reagent is used in an amount of generally1–1.5 equivalents, preferably 1.1–1.3 equivalents, relative to thecompound to be protected. Similarly, the base is used in an amount ofgenerally 1–3 equivalents, preferably 1.5–2 equivalents.

The solvent to be used for the protection of amino group may be water,methanol, ethanol, tetrahydrofuran, ethyl acetate, dichloromethane,chloroform, toluene and the like, a mixed solvent of these and the like,and a suitable solvent can be used depending on the reagent.

Examples of the base include pyridine, triethylamine, sodium hydroxide,potassium hydroxide, sodium carbonate, potassium carbonate, sodiumhydrogen carbonate, potassium hydrogen carbonate, disodiumhydrogenphosphate, dipotassium hydrogenphosphate and the like. For thecompound of the formula (1), sodium hydroxide and potassium hydroxideare particularly preferable, and for the compound of the formula (30),sodium hydrogen carbonate and potassium hydrogen carbonate arepreferable.

The reaction time varies depending on the reagent to be used and thereaction temperature. When t-butoxycarbonylation is applied usingdi-t-butyldicarbonate, the reaction ends in several minutes to about 2 hat 40° C. and several minutes to about 10 h at room temperature.

The carboxyl group can be generally protected by esterification. In thecase of a compound of the formula (1), for example, alcohol and: thionylchloride are mixed in advance and the compound of the formula (1) isadded for reaction. In the case of a compound of the formula (31), forexample, an esterifying agent, such as dimethylformamide dimethylacetaland the like, is used or a base and an alkylating agent are used toesterify carboxyl group.

The solvent to be used for esterification of carboxyl group may bemethanol, ethanol, t-butanol, benzyl alcohol, toluene, tetrahydrofuran,dichloromethane and the like or a mixed solvent of these and the like. Asuitable solvent can be used according to the ester group to beintroduced.

When the carboxyl group of the compound of the formula (1) isesterified, for example, generally, 2–3 equivalents of hydrogen chloridegas, 1–2 equivalents of acetyl chloride, or 1–2 equivalents ofp-toluenesulfonic acid is added into alcohol (for example, methanol togive methyl ester) to introduce an ester group.

Alternatively, alcohol and about 1–1.2 equivalents of thionyl chlorideare reacted and a compound of the formula (1) is added. While thereaction time varies depending on the reagent to be used, reactiontemperature and the like, when methanol-thionyl chloride is used formethyl esterification, for example, the reaction completes in generallyabout 3–5 h at 60° C.

In the case of a compound of the formula (31), for example, about1.5–2.5 equivalents of esterifying agent such as dimethylformamidedimethylacetal and the like is used for esterification. It is alsopossible to use a method wherein about 1–1.5 equivalents of alkyl halide(for example, methyl iodide and benzyl bromide) is reacted in thepresence of about 1–1.5 equivalents of a base (for example, amine suchas cyclohexylamine and the like, alkali metal salt such as cesiumcarbonate and the like) for protection. While the reaction time variesdepending on the reagent to be used and the reaction temperature, whenmethyl esterification is conducted in dichloromethane usingdimethylformamide dimethylacetal, the reaction completes in generallyabout 20–24 h at room temperature.

A step for producing optically active 6,6-dimethyl lysine derivative ofthe formula (3) or a salt thereof by reducing the nitro group of theoptically active amino acid derivative of the formula (2) is explainedin the following.

The nitro group can be reduced in a suitable alcohol solvent, such asmethanol, ethanol, isopropyl alcohol and the like or a mixed solvent ofsuch alcohol and water, preferably by [1] reduction using a combinationof metal and metallic sulfate or metallic chloride; [2] catalyticreduction using a combination of a transition metal catalyst andmetallic sulfate or metallic chloride; or [3] catalytic reduction usinga transition metal catalyst.

The [1] reduction using a combination of metal and metallic sulfate ormetallic chloride is explained in the following.

A preferable metal includes iron, zinc and the like. A preferablemetallic sulfate includes ferrous sulfate, copper sulfate, sodiumhydrogensulfate and the like. A preferable metallic chloride includeszinc chloride, cobalt chloride and the like. The metallic sulfate andmetallic chloride may be mixed for use.

A particularly preferable combination is iron and ferrous sulfate and/orsodium hydrogensulfate.

For example, the reduction can be conducted by the use of 1–40equivalents of iron and 1–20 equivalents of ferrous sulfate (and/orsodium hydrogensulfate), preferably 20–30 equivalents of iron and 10–15equivalents of ferrous sulfate (and/or sodium hydrogensulfate), relativeto the optically active amino acid derivative of the formula (2) andstirring them in the above-mentioned solvent preferably at a temperatureof from room temperature to 50° C. The pressure may be the atmosphericpressure. The time necessary for the reduction varies depending on themetal, metallic sulfate and the like to be added, but it is generally6–72 h.

The [2] catalytic reduction using a combination of a transition metalcatalyst and metallic sulfate or metallic chloride is explained in thefollowing.

A preferable transition metal catalyst is palladium catalyst, platinumcatalyst and the like. Such transition metal catalyst can be used in theform of palladium-carbon, platinum hydroxide, platinum dioxide,platinum-carbon and the like.

A preferable metallic sulfate is ferrous sulfate, copper sulfate and thelike. A preferable metallic chloride is zinc chloride, cobalt chlorideand the like. The metallic sulfate and metallic chloride may be mixedfor use.

A particularly preferable combination is palladium catalyst and ferroussulfate.

For example, the reduction can be conducted by the use of generally0.5–10 mol %, preferably 1–3 mol %, of a palladium catalyst andgenerally 1–10 equivalents of ferrous sulfate, preferably 2–5equivalents of iron, relative to the optically active amino acidderivative of the formula (2) and stirring them in the above-mentionedsolvent at a hydrogen pressure of generally 1–20 atm, preferably 1–5atm, at a reaction temperature of generally from room temperature to100° C., preferably from room temperature to 40° C. The time necessaryfor the reduction varies depending on the catalyst to be added,temperature, pressure and the like, but it is generally 3–48 h.

The [3] catalytic reduction using a transition metal catalyst isexplained in the following.

A preferable transition metal catalyst is palladium catalyst, platinumcatalyst and the like. These transition metal catalysts can be used inthe form of palladium-carbon, platinum hydroxide, platinum dioxide,platinum-carbon and the like.

For example, the reduction can be conducted by the use of generally0.5–10 mol %, preferably 1–3 mol %, of a palladium catalyst relative tothe optically active amino acid derivative of the formula (2) andstirring them in the above-mentioned solvent at a hydrogen pressure ofgenerally 10–30 atm, preferably 15–20 atm, at a reaction temperature ofgenerally 50° C.–100° C., preferably 70° C.–90° C. The time necessaryfor the reduction varies depending on the catalyst to be added and thelike, but it is generally 3–48 h.

The reduction [1] can be conducted at a relatively low temperature andlow pressure (for example, ordinary temperature, atmospheric pressure)but requires a relatively large amount of metal such as iron and thelike. The amount of the transition metal to be used for reduction [3] isrelatively small but the reduction requires reaction at a relativelyhigh temperature and a high pressure. The reduction [2] requiresrelatively small amounts of the transition metal and the like to be usedand can be conducted at a relatively low temperature and a low pressure(for example, ordinary temperature, atmospheric pressure), andtherefore, industrially superior to the above-mentioned methods [1] and[3]

The reaction mixture containing the thus-obtained optically active6,6-dimethyl lysine derivative of the formula (3) is concentrated (orevaporated) by concentration under reduced pressure and the like, andadjusted to about pH 8–9 with water and a base, such as sodiumcarbonate, sodium hydrogen carbonate and the like, and extracted with anorganic solvent, such as ethyl acetate, toluene, dichloromethane and thelike, or a mixed solvent, such as toluene-isopropyl alcohol and thelike.

The organic solvent layer is evaporated by concentration under reducedpressure and the like to give an optically active 6,6-dimethyl lysinederivative. The derivative can be further purified by a conventionalmethod such as chromatography and the like.

A step for producing an optically active lysine derivative of theformula (5) or a salt thereof by reacting an optically active6,6-dimethyl lysine derivative of the formula (3) or a salt thereof andan acetate derivative of the formula (4) is explained in the following.

The acetate derivative of the formula (4) can be synthesized easily froman α-hydroxyacetate derivative. For example, trifluoromethanesulfonicanhydride is added to benzyl α-hydroxyacetate in dichloromethane in thepresence of pyridine to give benzyl trifluoromethanesulfonyloxyacetate(Angewandte Chemie 1986, 98, 264). The α-halogenoacetate derivative ison the market from Sigma-Aldrich Japan K.K. and the like and can beobtained easily.

The 6,6-dimethyl lysine derivative of the formula (3) is dissolved in asuitable solvent and reacted with acetate derivative in the presence ofa base. The solvent is exemplified by acetonitrile, methanol, ethanol,isopropyl alcohol, ethyl acetate, methyl t-butyl ether, toluene and thelike. As the base, tertiary amine, such as triethylamine,diethylisopropylamine, diisopropylethylamine and the like, is used in anamount of generally 2–10 equivalents, preferably 3–4 equivalentsrelative to the 6,6-dimethyl lysine derivative. The acetate derivativeis used in an amount of generally 1–2 equivalents, preferably about 1.2equivalents. The mixture is stirred generally at 0° C.–40° C.,preferably at room temperature, for generally about 12–48 h, preferablyabout 18–24 h. The reaction mixture is concentrated (or evaporated) byconcentration under reduced pressure and the like, and extracted with anorganic solvent such as ethyl acetate, toluene, dichloromethane and thelike or a mixed solvent such as toluene-isopropyl alcohol and the like.Then, the organic solvent layer is evaporated under reduced pressure togive the optically active lysine derivative of the formula (5).

Alternatively, the optically active lysine derivative of the formula (5)is dissolved in a solvent such as tetrahydrofuran, dichloromethane,chloroform, acetone, acetonitrile and the like, to which are added anacid such as methanesulfonic acid, p-toluenesulfonic acid and the likein a proportion of generally 0.8–1.5 equivalents, preferably 1.0–1.1equivalents, and a poor solvent, such as ethyl acetate, diethyl ether,petroleum ether, methyl tert-butyl ether, tert-butyl acetate, isopropylacetate, hexane, cyclohexane, methylcyclohexane, heptane, toluene,xylene, methyl isobutyl ketone and the like, to allow precipitation(crystallization) of a salt, which salt is separated and dried to givecrystals of a salt of the optically active lysine derivative of theformula (5).

Alternatively, the optically active lysine derivative of the formula (5)is dissolved in a solvent such as ethyl acetate, diethyl ether,petroleum ether, methyl tert-butyl ether, tert-butyl acetate, isopropylacetate, hexane, cyclohexane, methylcyclohexane, heptane, toluene,xylene, methyl isobutyl ketone and the like, to which is added an acidsuch as methanesulfonic acid, p-toluenesulfonic acid and the like in aproportion of generally 0.8–1.5 equivalents, preferably 1.0–1.1equivalents, to allow precipitation (crystallization) of a salt, whichsalt is separated and dried to give crystals of a salt of the opticallyactive lysine derivative of the formula (5).

The present invention is explained in detail by referring to theexamples. The present invention is not limited by these examples in anyway.

REFERENCE EXAMPLE 1 methyl 4-methyl-4-nitropentanoate

2-Nitropropane (25 g, 0.28 mol) was dissolved in ethanol (140 ml) andmethyl acrylate (25.3 ml, 0.28 mol) and potassium fluoride (1.63 g,0.028 mol) were added. The mixture was refluxed under heating for 4 h.After cooling, ethanol was evaporated by concentration under reducedpressure, and the residue was extracted with ethyl acetate (100 ml) andwater (50 ml). Ethyl acetate was evaporated under reduced pressure togive the objective compound (40.6 g, yield 82.5%).

¹H-NMR(CDCl₃) δ ppm: 1.60(s,6H), 2.22–2.38(m,4H), 3.70(s,3H)

REFERENCE EXAMPLE 2 4-methyl-4-nitropentanol

Methyl 4-methyl-4-nitropentanoate (17.5 g, 0.1 mol) was dissolved inethanol (200 ml) and cooled to 5° C., after which sodium borohydride(7.55 g, 0.2 mol) was added. The mixture was stirred at room temperaturefor 1 h, and at 50° C. for 3 h. Water (100 ml) and hydrochloric acidwere added to adjust the pH to 3, and ethanol was evaporated byconcentration under reduced pressure. The residue was extracted withethyl acetate (250 ml) and water (100 ml) and washed with saturatedbrine. Ethyl acetate was evaporated by concentration under reducedpressure to give the objective compound (14.7 g, yield 91.2%).

¹H-NMR(CDCl₃) δ ppm: 1.5(m,2H), 1.61(s,6H), 1.95–2.05(m,2H), 3.65(t,2H)

REFERENCE EXAMPLE 3 4-methyl-4-nitropentanol methanesulfonate

4-Methyl-4-nitropentanol (4.3 g, 29.2 mmol) was dissolved in methylenechloride (100 ml) and triethylamine (6.1 ml, 43.8 mmol) was added, whichwas followed by ice-cooling. Methanesulfonyl chloride (2.94 ml, 38.0mmol) was added and the mixture was stirred for 1 h under ice-cooling.The mixture was washed with water (50 ml), 0.5 mol/L hydrochloric acid(50 ml), 5% aqueous sodium hydrogencarbonate solution (50 ml) andsaturated brine (50 ml). Methylene chloride was evaporated byconcentration under reduced pressure and the residue was subjected tocrystallization with ethyl acetate (4 ml) and n-hexane (20 ml). Afterfiltration, the residue was dried under reduced pressure at 40° C. togive the objective compound (6.17 g, yield 93.8%).

¹H-NMR(CDCl₃) δ ppm: 1.62(s,6H), 1.70–1.80(m,2H), 2.05(dd,2H),3.06(s,3H), 4.23(t,2H) mass spectrum m/e: 243(M+NH₄ ⁺)

EXAMPLE 1 4-methyl-4-nitropentyl iodide

4-Methyl-4-nitropentanol methanesulfonate (3.38 g, 15 mmol) wasdissolved in acetone (50 ml) and sodium iodide (8.7 g, 58 mmol) wasadded. The mixture was stirred overnight. Water (20 ml) was added andacetone was evaporated by concentration under reduced pressure. Theresidue was extracted with ethyl acetate (50 ml). The extract was washedwith 5% aqueous sodium thiosulfate solution (20 ml) and saturated brine(20 ml), and ethyl acetate was evaporated by concentration under reducedpressure to give the objective compound (3.84 g, yield 99.6%).

¹H-NMR(CDCl₃) δ ppm: 1.60(s,6H), 1.74–1.86(m,2H), 2.02(dd,2H),3.18(t,2H)

EXAMPLE 2 diethyl 2-acetylamino-2-(4-methyl-4-nitropentyl)malonate

20% Sodium ethoxide (3.74 g, 11 mmol) and diethyl 2-acetamidomalonate(2.39 g, 11 mmol) were dissolved in ethanol (10 ml). A solution of4-methyl-4-nitropentyl iodide (2.57 g, 10 mmol) in ethanol (8 ml) wasadded and the mixture was refluxed under heating for 5 h. Ethanol wasevaporated by concentration under reduced pressure and ethyl acetate (50ml) was added. The mixture was washed with water (20 ml) and saturatedbrine (20 ml). Ethyl acetate was evaporated by concentration underreduced pressure and the residue was purified by silica gel columnchromatography to give the objective compound (2.89 g, yield 83.4%).

¹H-NMR(CDCl₃) δ ppm: 1.00–1.14(m,2H), 1.25(t,6H), 1.54(s,6H),1.89(dd,2H), 2.05(s,3H), 2.33(dd,2H), 1.44(q,4H) mass spectrum m/e:347(MH⁺)

EXAMPLE 3 2-acetylamino-6-methyl-6-nitroheptanoic acid

Diethyl 2-acetylamino-2-(4-methyl-4-nitropentyl)malonate (193.5 g, 558.5mmol) was dissolved in ethanol (270 ml), and potassium hydroxide (51 g,781.9 mmol) was dissolved in water (250 ml) and added. The mixture wasstirred at 90° C. for 1 h, and potassium hydroxide (51 g, 781.9 mmol)was dissolved in water (250 ml) and added. The mixture was stirred atthe same temperature for 2 h. The mixture was cooled to 40° C. andconcentrated hydrochloric acid was added to adjust the solution to pH1.5, which solution was stirred overnight at 80° C. Ethanol wasevaporated by concentration under reduced pressure and the residue wasice-cooled. The precipitated crystals were separated, and the obtainedslurry was washed with ethyl acetate (200 ml) and dried under reducedpressure to give the objective compound (99.6 g, yield 72%).

¹H-NMR(CDCl₃) δ ppm: 1.23–1.44(m,2H), 1.57(s,6H), 1.66–1.78(m,2H),1.91(t,2H), 2.04(s,3H), 4.53(dd,1H) mass spectrum m/e: 245(MH⁻)

EXAMPLE 4 (S)-2-amino-6-methyl-6-nitroheptanoic acid

2-Acetylamino-6-methyl-6-nitroheptanoic acid (49.5 g, 200 mmol) wasdissolved in water (246 ml) and 30% sodium hydroxide was added to adjustits pH to 8.87. L-Acylase (4.95 g) and anhydrous cobalt chloride (0.27g) were added to adjust its pH to 9 and the mixture was stirredovernight at room temperature. Concentrated hydrochloric acid was addedto adjust its pH to 1.5 and the mixture was ice-cooled. The precipitatedcrystals were separated by filtration and the filtrate was washed twicewith a mixture (250 ml) of toluene-isopropyl alcohol (1:1) and theaqueous layer was concentrated under reduced pressure. Methanol (100 ml)was added for concentration and methanol (100 ml) was further added forconcentration. As a result, the objective compound was obtained (20.5g).

¹H-NMR(DMSO-d₆) δ ppm: 1.19–1.42(m,2H), 1.54(s,6H), 1.68–1.76(m,2H),1.86(t,2H), 3.53(t,1H)

EXAMPLE 5 methyl (S)-2-amino-6-methyl-6-nitroheptanoate hydrochloride

(S)-2-Amino-6-methyl-6-nitroheptanoic acid (20.5 g) was dissolved inmethanol (177 ml) and thionyl chloride (7.34 ml) was gently addeddropwise under ice-cooling. The mixture was refluxed under heating for2.5 h, and methanol was concentrated under reduced pressure to give theobjective compound (21.9 g).

¹H-NMR(CDCl₃) δ ppm: 1.35–1.50(m,2H), 1.60(s,6H), 1.92–2.08(m,4H),3.82(s,3H), 4.18(m,1H)

EXAMPLE 6 methyl (S)-2-t-butoxycarbonylamino-6-methyl-6-nitroheptanoate

Methyl (S)-2-amino-6-methyl-6-nitroheptanoate hydrochloride (21.9 g) wasdissolved in methanol (88.5 ml) and water (40 ml) and the mixture wasadjusted to pH 7.5 with saturated aqueous sodium hydrogencarbonatesolution. di-tert-Butyl-dicarbonate (21.9 g, 100.4 mmol) was dissolvedin methanol (44 ml) and added, and the mixture was stirred at roomtemperature for 1 h and at 40° C. for 2 h. Methanol was evaporated byconcentration under reduced pressure and the residue was extracted withethyl acetate (400 ml). The extract was washed with 0.5 mol/lhydrochloric acid (100 ml), saturated aqueous sodium hydrogencarbonatesolution (100 ml) and saturated brine (100 ml) and dried over anhydrousmagnesium sulfate. The anhydrous magnesium sulfate was removed byfiltration and the residue was concentrated under reduced pressure togive the objective compound (25.7 g, yield 80.5%).

¹H-NMR(CDCl₃) δ ppm: 1.23–1.37(m,2H), 1.43(s,9H), 1.58–1.68(s+m,8H),1.92(t,2H), 3.75(s,3H), 4.30(br,1H), 5.02(br,1H) mass spectrum m/e:319(MH⁺)

EXAMPLE 7 (S)-2-t-butoxycarbonylamino-6-methyl-6-nitroheptanoic acid

(S)-2-Amino-6-methyl-6-nitroheptanoic acid (10.25 g, 50.2 mmol) wasdissolved in methanol (40 ml) and water (25 ml).di-tert-Butyl-dicarbonate (10.92 g, 50.2 mmol) was dissolved in methanol(20 ml) and added, and the mixture was stirred at 40° C. for 1 h.di-tert-Butyl-dicarbonate (5.46 g, 25.1 mmol) was dissolved in methanol(10 ml) and added, and the mixture was stirred at 40° C. for 2.5 h.Methanol was evaporated by concentration under reduced pressure andethyl acetate (200 ml) and 6N hydrochloric acid were added for pHadjustment to 2.0 and extraction. The mixture was washed with water (100ml) and saturated brine (100 ml) and dried over anhydrous magnesiumsulfate. The solvent was evaporated by concentration under reducedpressure to give the objective compound (7.66 g).

¹H-NMR(CDCl₃) δ ppm: 1.30–1.42(m,2H), 1.47(s,6H), 1.55(s,9H),1.63–1.78(m,2H), 1.88–1.98(m,2H), 4.33(br,1H), 5.00(br,1H)

EXAMPLE 8 methyl (S)-2-t-butoxycarbonylamino-6-methyl-6-nitroheptanoate

(S)-2-t-Butoxycarbonylamino-6-methyl-6-nitroheptanoic acid (3.90 g,12.81 mmol) was dissolved in dichloromethane (20 ml) anddimethylformamide dimethylacetal (2.6 ml, 19.22 mmol) was added. Themixture was stirred at room temperature for 20 h. Dimethylformamidedimethylacetal (1.74 ml, 12.8 mmol) was added and the mixture wasstirred for 4 h. The mixture was washed with 0.5N hydrochloric acid (30ml), saturated aqueous sodium hydrogencarbonate solution (30 ml) andsaturated brine (30 ml). The solvent was evaporated by concentrationunder reduced pressure to give the objective compound (2.53 g, yield62%).

EXAMPLE 9 Nα-t-butoxycarbonyl-6,6-dimethyl-L-lysine methyl ester

Methyl (S)-2-t-butoxycarbonylamino-6-methyl-6-nitroheptanoate (10.05 g,31.6 mmol) was dissolved in methanol (200 ml). Iron powder (8.81 g,157.8 mmol) and ferrous sulfate 7 hydrate (40.5 g, 145.6 mmol) wereadded and the mixture was stirred at 40° C. for 17 h. Since the startingmaterial remained, the insoluble matter was removed by filtration. Ironpowder (8.81 g, 157.8 mmol) and ferrous sulfate 7 hydrate (40.5 g, 145.6mmol) were added and the mixture was stirred at 40° C. for 7 h. Theinsoluble material was removed by filtration, and methanol was removedby concentration under reduced pressure. Ethyl acetate (300 ml), water(100 ml) and 10% aqueous sodium carbonate solution were added for pHadjustment to 9 and extraction. Ethyl acetate was evaporated byconcentration under reduced pressure to give the objective compound(5.01 g, yield 55.0%).

¹H-NMR(CDCl₃) δ ppm: 1.10(s,6H), 1.46(s,9H), 1.56–1.68(m,2H),1.72(m,2H), 3.75(s,3H), 4.30(br,1H), 5.05(br,1H) ¹³C-NMR(CDCl₃) δ ppm:19.30, 27.31, 28.74, 32.29, 42.82, 48.82, 51.24, 52.29, 78.89, 154.45,172.44 mass spectrum m/e: 289(MH⁺)

EXAMPLE 10 Nα-t-butoxycarbonyl-6,6-dimethyl-L-lysine methyl ester

Methyl (S)-2-t-butoxycarbonylamino-6-methyl-6-nitroheptanoate (500 mg,1.57 mmol) was dissolved in methanol (8 ml) and water (0.5 ml), and 5%palladium-carbon (219 mg, 50% wet) and ferrous sulfate 7 hydrate (1.32g, 4.75 mmol) were added. The mixture was stirred at a hydrogen pressureof 3 atm and room temperature for 3 h. Palladium-carbon was removed byfiltration and methanol was evaporated by concentration under reducedpressure. Ethyl acetate (50 ml), water (50 ml) and sodium carbonate wereadded for pH adjustment to 10 and extraction. After layer partitioning,the aqueous layer was extracted again with toluene-isopropyl alcohol(1:1, 100 ml). The organic layers were combined and the solvent wasevaporated by concentration under reduced pressure to give the objectivecompound (350 mg, yield 77.3%).

EXAMPLE 11 Nα-t-butoxycarbonyl-6,6-dimethyl-L-lysine methyl ester

Methyl (S)-2-t-butoxycarbonylamino-6-methyl-6-nitroheptanoate (300 mg,0.94 mmol) was dissolved in methanol (10 ml) and 5% palladium-carbon(0.15 g, 50% wet) was added. The mixture was stirred at a hydrogenpressure of 20 atm and 80° C. for 24 h. Palladium-carbon was removed byfiltration and methanol was evaporated by concentration under reducedpressure to give the objective compound (219 mg, yield 80.8%).

EXAMPLE 12Nα-t-butoxycarbonyl-Nε-t-butoxycarbonylmethyl-6,6-dimethyl-L-lysinemethyl ester methanesulfonate

Nα-t-Butoxycarbonyl-6,6-dimethyl-L-lysine methyl ester (1.0 g, 3.46mmol) was dissolved in acetonitrile (30 ml), and diisopropylethylamine(1.60 ml, 9.19 mmol) and bromoacetyl t-butyl ester (0.812 g, 4.15 mmol)were added. The mixture was stirred at room temperature for about 20 h.Acetonitrile was evaporated by concentration under reduced pressure andethyl acetate (30 ml) was added. The insoluble material was removed byfiltration, and the mixture was washed twice with water (15 ml) and oncewith 10% sodium carbonate (15 ml) and dried over anhydrous sodiumsulfate. Sodium sulfate was removed by filtration and methanesulfonicacid (0.2 ml) was added. The mixture was stirred at room temperature.The precipitated crystals were separated and dried under reducedpressure to give the objective compound (1.18 g, yield 68.5%).

¹H-NMR(DMSO-d₆) δ ppm: 1.22(s,6H), 1.25–1.43(m+s,11H),1.49–1.68(s+m,13H), 2.30(s,3H), 3.63(s,3H), 3.87(m,2H), 3.98(m,1H)¹³C-NMR(DMSO-d₆) δ ppm: 19.63, 22.37, 27.63, 28.14, 30.80, 36.60, 41.90,51.70, 53.16, 59.12, 78.23, 83.09, 155.58, 166.41, 173.06 mass spectrumm/e: 403(MH⁺)

REFERENCE EXAMPLE 4 4-methyl-4-nitrovaleronitrile

Acrylonitrile (6.0 g, 113 mmol), 2-nitropropane (12.0 g, 135 mmol) andhexadecyltrimethyl ammonium chloride (2.0 g) were stirred in a 0.1Naqueous sodium hydroxide solution (200 ml) overnight at roomtemperature. The layers were separated and the aqueous layer wasextracted twice with methylene chloride (50 ml). The combined organiclayer was washed with saturated brine (30 ml). The solvent wasevaporated and the residue was dried to give oily4-methyl-4-nitrovaleronitrile (10.4 g, 73 mmol, yield 64.6%).

¹H-NMR(CDCl₃) δ ppm: 1.65(s,6H), 2.28–4.46(m,4H)

REFERENC EXAMPLE 5 4-methyl-4-nitropentanal

4-Methyl-4-nitrovaleronitrile (10.4 g, 13 mmol) was dissolved inmethylene chloride (100 ml) and 1M diisobutyl aluminum hydride-hexanesolution (100 ml) was added dropwise at −50° C. The mixture was stirredfor 0.5 h, and water (8 ml) was added gradually, after which magnesiumsulfate (5.0 g) was added. The mixture was filtrated and 1N hydrochloricacid (50 ml) was added. The mixture was stirred for 0.5 h and the layerswere separated. The organic layer was washed with saturated brine andthe solvent was evaporated. The residue was dried to give oily4-methyl-4-nitropentanal (6.9 g, 48 mmol).

¹H-NMR(CDCl₃) δ ppm: 1.60(s,6H), 2.25(t,2H), 2.52(t,2H), 9.79(s,1H)

EXAMPLE 13 5-(4-methyl-4-nitropentylidene)hydantoin

Oily 4-methyl-4-nitropentanal (4.0 g, 28 mmol), hydantoin (4.0 g, 40mmol) and sodium carbonate (2.8 g, 20 mmol) were added to 50% aqueousacetonitrile (200 ml) and the mixture was refluxed with stirring for 3days. The reaction mixture was concentrated and extracted three timeswith ethyl acetate. The organic layer was concentrated to dryness togive 5-(4-methyl-4-nitropentylidene)hydantoin (3.15 g, 13.9 mmol, yield50.0%).

¹H-NMR(DMSO-d₆) δ ppm: 1.56(s,6H), 2.00(m,2H), 2.13(m,2H), 5.42(t,1H)mass spectrum m/e: 226(MH⁻)

EXAMPLE 14 5-(4-methyl-4-nitropentylidene)hydantoin

Water (70 ml) was added to hydantoin (20.8 g, 210 mmol) and sodiumcarbonate (7.2 g, 70 mmol) and the mixture was dissolved by heating to85° C. 4-Methyl-4-nitropentanal (20.0 g, 140 mmol) was dissolved inisopropyl alcohol (130 ml) and added. The mixture was refluxed underheating for 2 h and isopropyl alcohol was evaporated under reducedpressure. Water (20 ml) and concentrated hydrochloric acid were added toadjust its pH to 8. The precipitated crystals were separated and driedto give 5-(1-hydroxy-4-methyl-4-nitropentylidene)hydantoin (27.3 g).This compound (19.4 g, 78.9 mmol) was dissolved in pyridine (60 ml) andmethanesulfonyl chloride (7.9 ml, 102.6 mmol) was added underice-cooling. The mixture was stirred at room temperature for 3 h. Water(50 ml) was added and the mixture was extracted three times with ethylacetate (50 ml). The extract solutions were combined and washed 4 timeswith 1 mol hydrochloric acid (50 ml). After separation of layers, theorganic layer was concentrated to dryness to give5-(1-methanesulfonyloxy-4-methyl-4-nitropentylidene)hydantoin (20.5 g).To this compound (5.74 g, 17.8 mmol) were added tetrahydrofuran (80 ml)and 1,8-diazabicyclo[5.4.0]undec-7-ene (2.7 ml, 17.8 mmol) and themixture was stirred at room temperature for 4 h. Tetrahydrofuran wasconcentrated under reduced pressure and the residue was extracted withethyl acetate (50 ml) and 0.5 M hydrochloric acid (80 ml). Afterseparation of layers, ethyl acetate was concentrated to dryness to give5-(4-methyl-4-nitropentylidene)hydantoin (4.0 g, 17.6 mmol, yield61.6%).

EXAMPLE 15 5-(4-methyl-4-nitropentyl)hydantoin

To 5-(4-methyl-4-nitropentylidene)hydantoin (3.15 g, 13.9 mmol) wereadded methanol (60 ml) and water (10 ml), and the mixture was reduced bythe addition of palladium-carbon (2.2 g) and hydrogen. After thereaction, palladium-carbon was filtered off and the residue wasconcentrated to dryness. Decantation with water and dichloromethane gave5-(4-methyl-4-nitropentyl)hydantoin (1.1 g).

¹H-NMR(DMSO-d₆) δ ppm: 1.18–1.30(m,2H), 1.52(s,6H), 2.55–2.70(m,2H),2.83–2.90(m,2H), 3.96–4.01(m,1H) mass spectrum m/e: 228(MH⁻)

EXAMPLE 16 5-(4-methyl-4-nitropentyl)hydantoin

Methanol (200 ml) was added to 5-(4-methyl-4-nitropentylidene)hydantoin(25.85 g, 114 mmol) and 27% aqueous sodium hydroxide solution was addedto adjust the pH to 10 for dissolution. Palladium-carbon (1.1 g) andhydrogen were added for reduction. After the reaction, palladium-carbonwas filtered off and methanol was evaporated by concentration underreduced pressure. Water (50 ml) was added and the pH was adjusted to 2with concentrated hydrochloric acid. The precipitated crystals wereseparated and dried to give 5-(4-methyl-4-nitropentyl)hydantoin (19.02g, 83 mmol, yield 72.9%).

In the following Examples, 2-amino-6-methyl-6-nitroheptanoic acid wasquantitatively determined by high performance liquid chromatography(HPLC) using a column “Inertsil ODS-2” (φ4.6×250 mm) manufactured by GLSciences Inc. The analysis conditions were as follows.

-   mobile phase: 30 mM aqueous phosphoric acid-   solution/methanol=8/2(V/V)-   flow rate: 1.0 ml/min.-   column temperature: 50° C.-   detection: UV 210 nm

The optical purity of 2-amino-6-methyl-6-nitroheptanoic acid wasmeasured by high performance liquid chromatography using an opticalresolution column “CROWNPAK CR(+)” (φ4.6×250 mm) manufactured by DAICELCHEMICAL INDUSTRIES, LTD. The analysis conditions were as follows.

-   mobile phase: aqueous perchloric acid solution (pH    1.8)/acetonitrile=8/2(V/V)-   flow rate: 1.0 ml/min.-   column temperature: 50° C.-   detection: UV 210 nm

Under these conditions, fractional quantitation was performed at 4.4 minretention time for (R)-2-amino-6-methyl-6-nitroheptanoic acid and at 6.1min retention time for (S)-2-amino-6-methyl-6-nitroheptanoic acid.

EXAMPLE 17

A medium (pH 7.0) containing glucose 0.5%, ammonium sulfate 0.5%, yeastextract powder 1.0%, peptone 1.0%, KH₂PO₄ 0.1%, K₂HPO₄ 0.3%, MgSO₄.7H₂O0.05%, FeSO₄.7H₂O 0.001%, MnSO₄.5H₂O 0.001% andDL-5-indolylmethylhydantoin 0.25% was dispensed by 50 ml to a 500 mlSakaguchi flask, and sterilized at 120° C. for 10 min. After cooling,Microbacterium liquefaciens AJ3940 cultured on an agar plate containingglucose 0.5%, ammonium sulfate 0.5%, yeast extract powder 1.0% andpeptone 1.0% at 30° C. for 24 h was inoculated by one platinum loop andsubjected to aerobic shake culture at 30° C. for 20 h. Thereafter, itwas centrifuged (12,000 g, 10 min) and bacterial cells were collected,which were suspended in 100 mM Tris-hydrochloric acid buffer (pH 8.0, 40ml). The suspension was centrifuged again in the same manner to givewashed bacterial cells. To the washed bacterial cells was added the sameweight of the same Tris-hydrochloric acid buffer to prepare a bacterialcell suspension. DL-5-(4-Methyl-4-nitropentyl)hydantoin was weighed by100 mg and suspended in 16 ml of 100 mM Tris-hydrochloric acid buffer(pH 8.0) and used as a substrate solution. To the substrate solution wasadded 4 ml of the bacterial cell suspension and the mixture wasincubated at 37° C. The amount of 2-amino-6-methyl-6-nitroheptanoic acidgenerated in the reaction mixture at 6, 20, 30, 44, 68 and 96 hoursafter the start of the reaction is shown in Table 1.

TABLE 1 2-amino-6-methyl-6- nitroheptanoic acid Residual substrateReaction time (min) (g/dl) generated (g/dl)  0 0 0.50  6 0.02 0.45 200.04 0.38 30 0.09 0.27 44 0.15 0.20 68 0.20 0.17 96 0.33 0.07

EXAMPLE 18

In the same manner as in Example 17, the reaction mixture (70 ml) wasprepared and reacted for 120 h. After the reaction, the reaction mixture(containing 207 mg of 2-amino-6-methyl-6-nitroheptanoic acid) wasdiluted in water (200 ml) and centrifuged (12,000 g, 10 min) to removethe bacterial cells. Then, the supernatant was passed through a cationexchange resin column (AMBERLITE IR-120B, column diameter 2.6 cm×length20 cm) (flow rate 3 ml/min) to allow adsorption of2-amino-6-methyl-6-nitroheptanoic acid. The column was washed with 500ml of water and then 2% aqueous ammonia (300 ml) was passed through thecolumn (flow rate 3 ml/min) to elute 2-amino-6-methyl-6-nitroheptanoicacid. The eluate was concentrated under reduced pressure to 3 ml using arotary evaporator at 40° C. Acetone was dropwise added thereto to give2-amino-6-methyl-6-nitroheptanoic acid as an ammonium salt (dry weight130 mg). The obtained 2-amino-6-methyl-6-nitroheptanoic acid wassubjected to HPLC analysis using an optical resolution column, CROWNPAKCR(+). As a result, the obtained acid was found to be an L-enantiomer(S-enantiomer) having an optical purity of not less than 99% e.e.

EXAMPLE 19

Agrobacterium sp. AJ11220 strain cultured on an agar plate containingglucose 0.5%, ammonium sulfate 0.5%, yeast extract powder 1.0% andpeptone 1.0% at 30° C. for 24 h was plated on the entirety of an agarplate (pH 7.0) containing glucose 0.5%, ammonium sulfate 0.5%, yeastextract powder 1.0%, peptone 1.0%, KH₂PO₄ 0.1%, K₂HPO₄ 0.3%, MgSO₄.7H₂O0.05%, FeSO₄ .7H₂O 0.001%, MnSO₄.5H₂O 0.001% andDL-5-indolylmethylhydantoin 0.25% and aerobically cultured at 30° C. for24 h. The bacterial cell was scraped and suspended in the same weight of100 mM Tris-hydrochloric acid buffer (pH 8.0) to give a bacterial cellsuspension. Then, DL-5-(4-methyl-4-nitropentyl)hydantoin (100 mg) andsodium sulfite (50 mg) were weighed and added to 8 ml of 100 mMTris-hydrochloric acid buffer (pH 8.0), which was used as a substratesolution. To the substrate solution was added 2 ml of the bacterial cellsuspension and the mixture was incubated at 30° C. The amount of2-amino-6-methyl-6-nitroheptanoic acid generated in the reaction mixtureat 1.5, 3, 6 and 20 hours after the start of the reaction is shown inTable 2. The reaction mixture after 20 hours from the start of thereaction was diluted with water and bacterial cells were removed. Theresulting liquid was subjected to HPLC analysis using an opticalresolution column, CROWNPAK CR(+). As a result, the obtained acid wasfound to be a D-enantiomer (R-enantiomer) having an optical purity ofnot less than 94% e.e.

TABLE 2 2-amino-6-methyl-6- nitroheptanoic acid (g/dl) Reaction time(min) generated 0 0 1.5 0.12 3 0.18 6 0.28 20 0.42

EXAMPLE 20

A medium (pH 7.0) containing maltose 2%, ammonium sulfate 0.5%, yeastextract powder 1.0%, peptone 1.0%, KH₂PO₄ 0.1%, K₂HPO₄ 0.3%, MgSO₄.7H₂O0.05%, FeSO₄.7H₂O 0.001%, MnSO₄.5H₂O 0.001% and DL-5-isopropylhydantoin0.2% was dispensed by 50 ml to a 500 ml Sakaguchi flask, and sterilizedat 120° C. for 10 min. After cooling, Bacillus sp. AJ12299 cultured onan agar plate containing glucose 0.5%, ammonium sulfate 0.5%, yeastextract powder 1.0% and peptone 1.0% at 30° C. for 24 h was inoculatedby one platinum loop and subjected to aerobic shake culture at 30° C.for 20 h. Thereafter, it was centrifuged (12,000 g, 10 min) andbacterial cells were collected, which were suspended in 100 mM potassiumphosphate buffer (pH 7.0, 40 ml). The suspension was centrifuged againin the same manner to give washed bacterial cell. To the washedbacterial cell was added the same weight of the same potassium phosphatebuffer to prepare a bacterial cell suspension.DL-5-(4-Methyl-4-nitropentyl)hydantoin (1.25 g/dl), glucose (1.25 g/dl)and MgSO₄.7H₂O (0.05 g/dl) were added to 100 mM potassium phosphatebuffer (pH 7.0) and used as a substrate solution. To the substratesolution (4 ml) was added 1 ml of the bacterial cell suspension and themixture was aerobically incubated while shaking in a test tube at 30° C.for 48 h. After the reaction, concentrated hydrochloric acid (0.4 ml)and 300 mM NaNO₂ (0.2 ml) were added to the reaction mixture (2.0 ml)under ice-cooling and the mixture was stood at 4° C. for 24 h, wherebyN-carbamyl-2-amino-6-methyl-6-nitroheptanoic acid generated by thereaction using the bacterial cell was decarbamylated. The concentrationof 2-amino-6-methyl-6-nitroheptanoic acid generated in the reactionmixture after decarbamylation was measured to be 0.51 g/dl. In addition,optical activity was measured. As a result, it was found that thecompound was an L-enantiomer (S-isomer) having an optical purity of notless than 99% e.e.

REFERENCE EXAMPLE 6

It was clarified by re-identification that Flavobacterium sp. AJ3912strain and AJ3940 strain were Microbacterium liquefaciens (formerAureobacterium liquefaciens).

That is, a physiological test was conducted and analyzed againstBergey's Manual of Determinative Bacteriology, vol. 1 (9th Ed., 1994,Williams & Wilkins), which is a taxonomy book of bacteria. The followingphysiological properties were found.

test Gram stain positive mobility absent nitrate reduction −pyrimidinase − pyridonylallyl amidase − alkaline phosphatase +β-glucuronidase − β-galactosidase + α-glucosidase +N-acetyl-β-glucosaminidase + esculin (glucosidase) + urease − gelatinliquefaction + fermentability of carbohydrate glucose − ribose − xylose− mannitol − maltose − lactose − sucrose − glycogen − anaerobic growth −casein hydrolysis +

REFERENCE EXAMPLE 7

It was clarified by re-identification that the Pseudomonushydantoinophilum AJ11220 strain was Agrobacterium sp.

That is, a physiological test was conducted and analyzed againstBergey's Manual of Determinative Bacteriology, vol. 1 (9th Ed., 1994,Williams & Wilkins), which is a taxonomy book of bacteria. The followingphysiological properties were found.

test cell size and shape 0.8 × 1.5 − 2.0 μm Gram stain − spore −motility and flagellar arrangement +: peritrichous catalase + oxidase +O-F test − nitrate reduction + indole formation − acid production fromD-glucose − arginine dihydrolase reaction − urease + esculinhydrolysis + gelatin hydrolysis − β-galactosidase + utilization ofcarbon compounds glucose + L-arabinose + D-mannose + D-mannitol +N-acetyl-D-glucosamine + maltose + potassium gluconate − n-caprate −adipate − dl-malic acid + sodium citrate − phenylacetate −

According to the present invention, optically active lysine derivativesof the aforementioned formulas (3) and (5) useful as pharmaceuticalintermediates can be produced by an industrial method.

This application is based on patent application Nos. 213181/2000,334579/2000 and 118508/2001 filed in Japan, the contents of which arehereby incorporated by reference.

1. A compound of any of the formulas (14)–(17), a salt thereof, an optically active substance thereof or a racemate thereof:

wherein P₁ and P₂ are each independently an amino-protecting group or hydrogen atom where P₁ and P₂ are not hydrogen atoms at the same time, or P₁ and P₂ in combination show an amino-protecting group except phthaloyl group, P_(3a) is a carboxyl-protecting group and P₄ is a hydrogen atom or a carboxyl-protecting group.
 2. A compound of any of the formulas (19)–(22):

or a salt thereof.
 3. A compound of formula (24) or a salt thereof:

wherein R₄ is methyl group or phenyl group, and wherein formula (24) comprise its optically active substance and racemate. 